Naturally occurring regulatory T-cells (nTregs;CD4+CD25+Foxp3+) are known to suppress a wide range of immune responses via several different mechanisms including production of regulatory cytokines, competition for essential cytokines and contact-dependent mechanisms. Because nTregs have been shown to suppress Th1 and Th17 autoimmune responses in vivo, a great deal of interest has been generated regarding the possible use of these cells to treat patients with chronic inflammatory diseases such as Crohn's disease (CD). A major limitation in investigating the use of nTregs for the treatment of CD is the relative paucity of these suppressor cells as they constitute <10% of the peripheral CD4+ T-cell population in mice and 1-2% of the CD4+ T-cells in humans. In an attempt to circumvent this significant limitation, we have developed a novel ex vivo method that generates large numbers of Foxp3-expressing Tregs that can be used to evaluate their therapeutic efficacy in a mouse model of chronic colitis. We present preliminary data demonstrating that polyclonal activation of conventional CD4+Foxp3- in the presence of IL-2, TGF[unreadable] and all-trans retinoic acid induces >90% conversion of these T-cells to Foxp3-expressing Tregs (termed iTregs) as well as a 4-5-fold increase in proliferation following a 4 day incubation period in vitro. Furthermore, we find enhanced surface expression of the gut-homing adhesion molecule a4[unreadable]7 suggesting tissue-specific imprinting of iTregs by our conversion/expansion protocol. Finally, we provide preliminary data demonstrating that iTregs are significantly more potent at suppressing responder T-cell activation in vitro and equally effective as nTregs at reversing established colitis in vivo. The overall objective of this proposal is to identify where Tregs exert their suppressive activity during active gut inflammation and define how these lymphocytes traffic to these tissues. Hypothesis: We propose that ex vivo generated iTregs utilize CD62L (L-selectin) and/or a4[unreadable]7 to traffic to the inflamed Peyer's patches (PPs), mesenteric lymph nodes (MLNs) and/or colonic lamina propria (LP) where they suppress Th1/Th17 effector cell function thereby reversing/attenuating active disease. In order to test our hypothesis we propose to: a) Determine whether PPs and/or MLNs are required for iTreg-mediated reversal/suppression of preexisting colitis and b) Identify the specific T-cell-associated adhesion molecules that is/are required for iTreg-mediated attenuation of active colitis. Data obtained from these Exploratory/ Developmental (R21) studies will advance the field by providing new and important mechanistic information on the anatomic location(s) where Tregs suppress preexisting gut inflammation and will identify which of the different Treg adhesion molecules is/are required for homing to these tissues to suppress disease. In addition, the studies outlined in the current application will provide the necessary preliminary data that will be used to initiate a more detailed investigation (R01) exploring how ex vivo-generated Tregs may be manipulated to more specifically target them to the inflamed lymphoid tissue and gut. If Treg-based adoptive immunotherapy is to become a viable therapeutic approach for the treatment of CD (or ulcerative colitis), then it is crucial to better understand where and how these regulatory cells exert their protective effects in vivo. PUBLIC HEALTH RELEVANCE: The work proposed in this application is designed to improve our understanding of where and how ex vivo-generated regulatory T-cells (Tregs) attenuate preexisting gut inflammation. Data obtained from these studies may provide the necessary data to begin to develop a Treg-based therapeutic strategy for the treatment of patients with inflammatory bowel disease.